Mechanisms for establishing transcriptional competence of tissue-specific genes in pluripotent stem cells

Abstract

Tissue-specific genes have been found to be epigenetically marked in embryonic stem cells by different combination of histone modifications. These marks are believed to prime genes for activation at later stages of development. Previous work has revealed the presence of one such signature on one enhancer of the mouse λ5-VpreB1 locus, which is expressed at the pro- and pre-B cell stage of B lymphocyte development. This element, which has been called Early- Transcriptional Competence Mark (ETCM), is characterised by the presence of tightly localised peaks of histone H3K4 methylation and H3K9 acetylation. However, the ETCM lacks the trimethylation of H3K27 that defines bivalent domains at many tissue-specific genes in ES cells. The results described in this thesis show that that two ES cell transcription factors, Sox2 and Foxd3, bind to the λ5-VpreB1 ETCM in ES cells. Analysis of factor binding to the λ5-VpreB1 locus at the proB cell stage of B-cell development showed that the same sequences are occupied by the B-cell specific factors Sox4 and Foxp1. Chromatin immunoprecipitation and microarray analysis (ChIP-on-Chip) showed that Sox2 and Foxd3 are bound to a number of ETCM-like elements in ES cells, as well as bivalent domains, suggesting that they are involved in the establishment and maintenance of these epigenetic marks in ES cells. Knockdown of Sox2 by siRNA confirmed its involvement in the methylation of H3K4 on these DNA regions. Based on these data, I propose a factor-relay model in which embryonic stem cell transcription factors generate epigenetic signatures at key regulatory elements of tissue-specific genes. During differentiation these proteins are replaced by related factors, which share their DNA-binding profile, and play an important role in activating tissue-specific transcription.